Immunoassays can be used to quantitatively determine a concentration of target entities, for example antigens, present in a sample. In the case of microfluidic immunoassays, an arrangement comprising a microfluidic chamber into which a sample is introduced may be used. Such a chamber comprises a plurality of probe entities, for example antibodies, immobilized on a surface of the chamber such that, as the sample is passed over the surface, antigens in the sample bind to the antibodies in the chamber. The amount of antibody-antigen binding may be detected and quantified using, for example, fluorescence or surface plasmon resonance measurements (SPR) and a concentration of target entities in the sample can be determined from this amount.
Due to the constraints inherent in existing immunoassays, for example the probe density or detection sensitivity, the range of concentrations of target entities in a sample which can be detected by known microfluidic immunoassays is limited. For any one assay, detection and quantification will only work in a sensitive (ideally linear) range of the relationship between target concentration and detection signal. Below this range, signal to noise ratios are too low and above this range the assay saturates. In either case, the measured signal becomes independent of sample concentration.
It would be desirable to provide methods and systems for determining target concentration that address these issues and extend the dynamic range over which concentrations can be quantified.